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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2018 to 17 Jul 2018
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
other: ISO 14669
Version / remarks:
ISO International Standard 14669. "Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)", First edition 1999-04-01.
according to
other: OECD 23
Version / remarks:
Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000.
GLP compliance:
Screening study completed - o evaluate the test item for its ability to generate acute lethal toxic effects on marine copepod Tisbe battagliai during an exposure period of 48 hours.

Test material

Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity/Composition: 99.73%

Sampling and analysis

Analytical monitoring:
Details on sampling:
2.2.2. Test Samples
Test samples were stored in the freezer (£ -15°C). Storage stability of samples under these conditions was demonstrated in project 20141828.
On the day of analysis, the test samples were thawed at room temperature. The samples were diluted in a 9:1 (v:v) ratio with acetonitrile and analyzed. If necessary, the samples were further diluted with 10/90 (v/v) acetonitrile/natural seawater to obtain concentrations within the calibration range. All solutions containing the test item were protected from light.

2.2.3. Preparation of Solutions
All solutions containing the test item were protected from light.

Stock and Spiking Solutions
Stock solutions of the test item were prepared in acetonitrile at concentrations of 1000 and 2000 mg/L.
Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of the spiking solutions was acetonitrile.

Calibration Solutions
Six calibration solutions in the concentration range of 0.08 – 20 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was 10/90 (v/v) acetonitrile/natural seawater.

Quality Control (QC) Samples
4.5 mL blank medium was spiked with the test item at a target concentration of 0.1 or 100 mg/L. The QC samples were treated similarly as the test samples (see paragraph 2.2.2 Test Samples’). Blank QC samples consisting of blank medium were treated similarly to the QC and test samples.

2.2.4. Sample Injections
Calibration solutions were injected in duplicate. Test samples and QC samples were analyzed by single injection.

Test solutions

Details on test solutions: Test Concentrations
PF-06961030 Solutions containing 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L

Test organisms

Test organisms (species):
other aquatic crustacea: Tisbe battagliai (Copepoda, Crustacea) (Volkmann-Rocco),
Details on test organisms:
Source Guernsey Sea Farms Ltd., Guernsey, United Kingdom.
Reason for selection This system has been selected as an internationally accepted invertebrate species.
Characteristics 6 ± 2 days old copepodids

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

Test temperature:
18-22°C, constant within 2°C.
Between 6.0 and 8.5.
Dissolved oxygen:
dissolved oxygen concentration >80% of the air saturation value at the start of the test, >= 4 mg/L at the end of the test.
35‰ at start of test
Nominal and measured concentrations:
4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L.
Details on test conditions:
Test duration 48 hours
Test type Static
Test vessels 24-well plastic plates covered with a lid.
Volume of vessel 3 ml
Medium 10 μm filtered and UV treated natural marine water of the same origin as the water that was used for culturing the organisms. Water was obtained from Guernsey Sea Farms Ltd., Guernsey, United Kingdom and stored at 4ºC until test initiation. Specifications: dissolved oxygen concentration >80% of the air saturation value, pH = 8, Salinity = 35‰.
Number of copepodids 20 for each control and test item concentration
Loading 5 per vessel containing 2.5 ml of test solution
Light The study was performed in the dark (max. 1 lux)
Temperature 18-22°C, constant within 2°C.
Oxygen concentration >= 4 mg/L at the end of the test.
Aeration No aeration of the test solutions.
pH Between 6.0 and 8.5.
Feeding No feeding
Introduction of copepodids Within 53 minutes after preparation of the test solutions.
Reference substance (positive control):
Zinc sulfate heptahydrate

Results and discussion

Effect concentrationsopen allclose all
24 h
Dose descriptor:
Effect conc.:
12 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
11 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
The NOEC was determined with Fisher’s Exact Binomial Test with Bonfferoni Correction. The NOEC was determined to be 4.6 mg/L.
Results with reference substance (positive control):
At the end of the test, 50% mortality was observed in the reference control (see Table 2). Since the effect in this treatment should be between 20 and 80% it was concluded that the batch copepodids used showed normal sensitivity to zinc sulphate heptahydrate.

Applicant's summary and conclusion

Validity criteria fulfilled:
In conclusion, under the conditions of the present study with Tisbe battagliai the 48h-LC50 was 11 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 9.3 and 14 mg/L). The NOEC was determined to be 4.6 mg/L.